Bystander effects of bioreductive drugs: potential for exploiting pathological tumor hypoxia with dinitrobenzamide mustards

Tumor hypoxia is an important therapeutic target, and it can potentially be exploited by hypoxia-activated prodrugs. However, physiological hypoxia in normal tissues is a limitation. One solution would be to confine activation to severely (pathologically) hypoxic tissue, using hypoxia-activated prodrugs that provide a bystander effect through diffusion of the activated cytotoxin to adjacent regions at intermediate oxygen concentrations (associated with partial radioresistance). To evaluate this requirement, we identified five hypoxia-activated prodrugs with at least 10-fold higher potency against a cell line (A549-P540(puro)) overexpressing human cytochrome P450 reductase (P450R) relative to A549-Lo21 cells with 200-fold lower P450R activity. Bystander killing by these hypoxia-activated prodrugs was tested in anoxic multicellular layer co-cultures of these two cell lines. Cytotoxic potency against A549-Lo21 cells was unaffected by the presence of A549-P450(puro) cells for tirapazamine and RSU-1069 but increased more than 10-fold for the aziridinyldintrobenzamide CB 1954, more than 14-fold for the corresponding nitrogen mustard SN 23862, and 15-fold for its water-soluble analog SN 23816. The cytotoxic extracellular metabolites resulting from hypoxic nitroreduction of CB 1954 and SN 23862 by A549-P450(puro) cells were identified by LC/MS and bioassay methods. For SN 23862, these included the 2-amine metabolite, previously, identified as the bystander metabolite from aerobic activation by the E. coli nfsB nitroreductase, but also novel di-reduced metabolites. Cytotoxicity of SN 23862 to A549-P450(puro) cells was inhibited by lower concentrations of oxygen than for tirapazamine. The combination of selective activation under severe hypoxia with an efficient bystander effect identifies the dinitrobenzamide mustards for further development as hypoxia-activated prodrugs.     

Isophosphoramide mustard analogues as prodrugs for anticancer gene-directed enzyme-prodrug therapy (GDEPT)

Two types of prodrugs, benzyl analogues of isophosphoramide mustard (iPAM), activated by cytochrome P450, and acylthioethyl analogues, activated by esterases, were designed. In contrast to iPAM that hydrolyse rapidly, the examined compounds are stable in phosphate-buffered saline and Tris buffer. Benzyl analogues of iPAM are poor substrates for cytochrome P450, are not cytotoxic and posses no antitumour activity. Acylthioethyl analogues of iPAM are good substrates for pig liver esterase, are cytotoxic and exert antitumour activity against L1210 leukaemia in mice. The observed correlation for iPAM analogues between their susceptibility to hydrolysis and cytotoxicity and antitumour activity suggests possible application of these compounds as the prodrugs in gene-directed enzyme-prodrug therapy.     

Synthesis and cytotoxicity of pyranonaphthoquinone natural product analogues under bioreductive conditions

We have synthesised a focused library of derivatives of natural products containing the pyranonaphthoquinone moiety including the first report of such a scaffold with an appended tetrazole functionality. Examples include kalafungin derivatives as well as analogues of nanaomycin and eleutherin. These compounds were assessed for cytotoxic activation by breast cancer cell lines engineered to express the prototypic human one- and two-electron quinone bioreductive enzymes, NADPH: cytochrome P450 oxidoreductase (POR) and NAD(P)H: quinoneoxidoreductase 1 (NQO1; DT-diaphorase), respectively. Several compounds were observed to be cytotoxic at sub-micromolar level and a pattern of increased aerobic potency was observed in cells over expressing POR. A subset of analogues was assessed under anoxic conditions, where cytotoxicity was reduced, implicating redox cycling as a major mechanism of toxicity. The substrate specificity for reductive enzymes is relevant to the future design of bioreductive prodrugs to treat cancer.     

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO₂) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO₂) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.     

Hypoxia-Targeting Drug Evofosfamide (TH-302) Enhances Sunitinib Activity in Neuroblastoma Xenograft Models

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other.     

Regulation of cyclooxygenase-2 by hypoxia and peroxisome proliferators in the corneal epithelium

Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR alpha and beta but not gamma mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E(2) accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation.     

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b₅ on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.     

Evidence for requirement of NADPH-cytochrome P450 oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system

Rabbit antibodies raised against the hydrophilic part of microsomal NADPH-cytochrome P450 oxidoreductase (denoted fpT) demonstrated a marked ability to inhibit NADPH-sterol Delta7-reductase activity. In addition, trypsin and proteinase K treatment of microsomes removed almost all microsomal electron transfer constituents from the microsomes, but the Delta7-reductase activity could be reconstituted by adding detergent-solubilized NADPH-cytochrome P450 oxidoreductase (denoted OR). Furthermore, after solubilization from microsomes, the Delta7-reductase activity could be reconstituted with OR in a DEAE-cellulose column chromatography eluate fraction, which contained little OR activity. In the microsomal system, carbon monoxide, ketoconazole, and miconazole, specific inhibitors of cytochrome P450, had no effect on Delta7-reductase activity. These results provide the first evidence of an essential requirement of OR, which is distinct from cytochrome P450, in the NADPH-sterol Delta7-reductase system. EDTA, o-phenanthroline and KCN markedly lowered Delta7-reductase activity in a dose-dependent manner. Among metal ions tested, only ferric ion restored the reductase activity in the EDTA-treated microsomes. These results sugguest that NADPH-sterol Delta7-reductase is membrane-bound iron-dependent protein embedded in the microsomal lipid bilayer.     

Design,synthesis and biological evaluation of 2H-benzo[b][1,4] oxazine derivatives as hypoxia targeted compounds for cancer therapeutics

A small library of 2H-benzo[b][1,4] oxazine derivative was synthesized and their biological activity was tested on HepG2 cells under normoxic and hypoxic conditions. From preliminary screening, we found compound 10 and 11 specifically inhibit hypoxic cancer cell growth IC₅₀ 87 ± 1.8 μM and IC₅₀ 10 ± 3.7 μM while sparing ‘normoxic’ cells IC₅₀ >600 M and >1 mM (not applicable), respectively. We tested the effect of 10 on MTT, clonogenic and hypoxia induced genes. The MTT correlates with clonogenic assays and most importantly compound 10 down regulates hypoxia induces genes (HIF-1α, P21 and VEGF) appropriately. We are in the process to explore the molecular mechanism of action of oxazine derivative compounds on hypoxia tumor cells.     

Effects of a transition from normoxia to anoxia on yeast cytochrome c oxidase and the mitochondrial respiratory chain: Implications for hypoxic gene induction

Previous studies have demonstrated that the mitochondrial respiratory chain and cytochrome c oxidase participate in oxygen sensing and the induction of some hypoxic nuclear genes in eukaryotes. In addition, it has been proposed that mitochondrially-generated reactive oxygen and nitrogen species function as signals in a signaling pathway for the induction of hypoxic genes. To gain insight concerning this pathway, we have looked at changes in the functionality of the yeast respiratory chain as cells experience a shift from normoxia to anoxia. These studies have revealed that yeast cells retain the ability to respire at normoxic levels for up to 4 h after a shift and that the mitochondrial cytochrome levels drop rapidly to 30-50% of their normoxic levels and the turnover rate of cytochrome c oxidase (COX) increases during this shift. The increase in COX turnover rate cannot be explained by replacing the aerobic isoform, Va, of cytochrome c oxidase subunit V with the more active hypoxic isoform, Vb. We have also found that mitochondria retain the ability to respire, albeit at reduced levels, in anoxic cells, indicating that yeast cells maintain a functional mitochondrial respiratory chain in the absence of oxygen. This raises the intriguing possibility that the mitochondrial respiratory chain has a previously unexplored role in anoxic cells and may function with an alternative electron acceptor when oxygen is unavailable.     

Noncompetitive mixed-type inhibition of rainbow trout CYP1A catalytic activity by clotrimazole

Over the past two decades a number of antifungal imidazole derivatives have been approved for use in agricultural. The purpose of this study was to characterize the interaction of a model antifungal imidazole compound with a cytochrome P450 isozyme in a species of fish. Clotrimazole inhibited rainbow trout (Oncorhyncus mykiss) hepatic CYP1A-catalyzed ethoxyresorufin O-deethylase (EROD) activity in vivo and in vitro. Although clotrimazole inhibited EROD activity in vivo, it did not effect CYP1A mRNA levels. Addition of clotrimazole to microsomes produced a type II binding spectrum and clotrimazole was determined to be a noncompetitive mixed-type inhibitor of EROD activity with an IC₅₀ of 190 nM. Since antifungal imidazole compounds may be co-applied with other pesticides, inhibition of cytochrome P450 activity by antifungal imidazole compounds may lead to unexpected toxicological interactions.     

Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations

Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.     

Tri1 in Fusarium graminearum Encodes a P450 Oxygenase

Gibberella zeae (asexual state Fusarium graminearum) is a major causal agent of wheat head blight and maize ear rot in North America and is responsible for contamination of grain with deoxynivalenol and related trichothecene mycotoxins. To identify additional trichothecene biosynthetic genes, cDNA libraries were prepared from fungal cultures under trichothecene-inducing conditions in culture and in planta. A gene designated LH1 that was highly expressed under these conditions exhibited only moderate (59%) similarity to known trichothecene biosynthetic cytochrome P450s. To determine the function of LH1, gene disruptants were produced and assessed for trichothecene production. Gene disruptants no longer produced 15-acetyldeoxynivalenol, which is oxygenated at carbon 7 (C-7) and C-8, but rather accumulated calonectrin and 3-deacetylcalonectrin, which are not oxygenated at either C-7 or C-8. These results indicate that gene LH1 encodes a cytochrome P450 responsible for oxygenation at one or both of these positions. Despite the relatively low level of DNA and amino acid sequence similarity between the two genes, LH1 from G. zeae is the probable homologue of Tri1, which encodes a cytochrome P450 required for C-8 oxygenation in F. sporotrichioides.     

Semisynthesis,cytotoxicity, antiviral activity,and drug interaction liability of 7-O-methylated analogues of flavonolignans from milk thistle

Silymarin, an extract of the seeds of milk thistle (Silybum marianum), is used as an herbal remedy, particularly for hepatoprotection. The main chemical constituents in silymarin are seven flavonolignans. Recent studies explored the non-selective methylation of one flavonolignan, silybin B, and then tested those analogues for cytotoxicity and inhibition of both cytochrome P450 (CYP) 2C9 activity in human liver microsomes and hepatitis C virus infection in a human hepatoma (Huh7.5.1) cell line. In general, enhanced bioactivity was observed with the analogues. To further probe the biological consequences of methylation of the seven major flavonolignans, a series of 7-O-methylflavonolignans were generated. Optimization of the reaction conditions permitted selective methylation at the phenol in the 7-position in the presence of each metabolite’s 4–5 other phenolic and/or alcoholic positions without the use of protecting groups. These 7-O-methylated analogues, in parallel with the corresponding parent compounds, were evaluated for cytotoxicity against Huh7.5.1 cells; in all cases the monomethylated analogues were more cytotoxic than the parent compounds. Moreover, parent compounds that were relatively non-toxic and inactive or weak inhibitors of hepatitis C virus infection had enhanced cytotoxicity and anti-HCV activity upon 7-O-methylation. Also, the compounds were tested for inhibition of major drug metabolizing enzymes (CYP2C9, CYP3A4/5, UDP-glucuronsyltransferases) in pooled human liver or intestinal microsomes. Methylation of flavonolignans differentially modified inhibitory potency, with compounds demonstrating both increased and decreased potency depending upon the compound tested and the enzyme system investigated. In total, these data indicated that monomethylation modulates the cytotoxic, antiviral, and drug interaction potential of silymarin flavonolignans.     

Hepatic microsomal alterations during Dipetalonema viteae infection in Mastomys natalensis

The multimammate rat, Mastomys natalensis was used as a model system to evaluate the chronic effects of infection by Dipetalonema viteae on hepatic mixed function oxidase activity. Total hepatic cytochrome P450 content and related total tissue mixed function oxidase activity were decreased to about 50% of control levels at patent phase of infection. The decrease in total tissue mixed function oxidase activity was due to a large decrease in cytochrome P450 concentration in the endoplasmic reticulum. Although the decrease in total liver monooxygenase activity in two substrates aniline and aminopyrine roughly paralleled the loss in cytochrome P450 content, several other microsomal enzyme markers not related to cytochrome P450 monooxygenation were elevated in proportion to total liver microsomal protein content. These results suggest that in M. natalensis during experimental filariasis, there is proliferation of hepatic cells with normal content of endoplasmic reticulum. Furthermore, there appears to be selective toxicity for hepatic cytochrome P450 and related monooxygenase activities. This may compromise the animal's ability to metabolize and dispose of other drugs to which the animals may be exposed in the course of infection.     

NADPH-cytochrome P450 reductase.

The rate of reduction of cytochrome P450 in hepatic microsomes in the presence of NADPH has been measured with a dual wavelength stopped-flow spectrophotometer. The results obtained, with microsomes prepared from phenobarbital-pretreated rats, indicate that the reduction process is biphasic and most probably composed of two concurrent first-order reactions. The rate constant for the reduction of cytochrome P450 in the fast phase in the presence of ethylmorphine is 1.74 s^?1. Since approximately 50% or more of the cytochrome P450 is reduced in the fast phase under these conditions, the rate of reduction of cytochrome P450 is approximately 150 nmol min^?1 (mg of protein)^?1. Under similar conditions the rate of ethylmorphine N-demethylation is 8.6 nmol min^?1 (mg of protein)^?1. Thus the rate-limiting step in ethylmorphine N-demethylation cannot be the introduction of the first electron into cytochrome P450 by NADPH-cytochrome P450 reductase.     

Xenobiotic metabolism in helminths

During its life cycle a parasite, like mammals and other organisms, comes into contact with a variety of toxic molecules. In mammals the main line of defence against such compounds is a group of oxidative enzymes that occur predominantly in the liver. Many of these oxidations are dependent on the haemoprotein cytochrome P-450 which serves as a terminal oxidase accepting electrons from NADPH and cytochrome [Formula: see text] . This review by Wendy Precious and John Barrett illustrates that in contrast to mammals, xenobiotic metabolism in both parasitic and free-living platyhelminths and nematodes is predominantly reductive and hydrolytic as the cytochrome P-450 system is absent. This is surprising since it is present in many groups including bacteria, fungi and protozoa, which suggests an ancient origin. The absence of the cytochrome P-450 system not only severely limits the ability of helminths to detoxify compounds but also limits their ability to activate prodrugs.     

Genetic analysis of aflatoxin B1 activation in rat hepatoma cells

Summary We present a strategy to elucidate the rate-limiting steps in activation of carcinogenic compounds by cytochromes P450. The principle was to select Reuber rat hepatoma cells for resistance to a procarcinogen. The hypothesis was that resistant cells should be systematically deficient in the P450 enzyme(s) involved in the activation process. Here we present an example of the use of this approach using aflatoxin B1 (AFB1), a potent hepatocarcinogen, as the selective agent. Parental cells as well as individual and pooled colonies selected for AFB1 resistance from three independent rat hepatoma lines were characterized for their content of 1) mRNA hybridizing to cDNA and/or oligonucleotide probes for cytochromes P450IIB1, P450IIB2 and albumin; and 2) aldrin epoxidase activity. Parental aflatoxin B1-sensitive cells were shown to express P450IIB1 but not P450IIB2. The majority of the aflatoxin B1-resistant clones failed to accumulate cytochrome P450IIB1 mRNA and expressed no or only very low aldrin epoxidase activity. Albumin mRNA levels remained unchanged, demonstrating that loss of expression of cytochrome P450IIB1 was not a consequence of a general dedifferentiation event. A revertant population showing restoration of both cytochrome P450IIB1 mRNA accumulation and aldrin epoxidase activity was fully sensitive to aflatoxin B1. The correlation between expression of cytochrome P450IIB1 and sensitivity to aflatoxin B1 in both parental cells and revertants strongly suggests that cytochrome P450IIB1 is a major contributor to the activation of aflatoxin B1 in rat hepatoma cells. The kind of strategy described here could be applied to other compounds that become cytotoxic for hepatoma cells following activation by cytochromes P450.Abbreviations AFB1 aflatoxin B1 - AE aldrin epoxidase - AHH aryl hydrocarbon hydroxylase - PAH polycyclic aromatic hydrocarbons - PB phenobarbital